Abstract: Quantitative PCR technology has become the most commonly used method for the gene expression level determination. Choosing the appropriate reference gene is very important for the accurate quantitative gene to gene expression level. In this paper, the expressions of four internal reference genes，including β-action、GAPDH、EF1-α和18S rRDA, in different tissues, growth environments and development stages of T. flavidus were detected, and the stability of the expression of these four internal reference genes was comprehensively analyzed by using three internal reference gene screening software ( geNorm, NormFinder and Bestkeeper) . The results showed that the stable rank of the internal reference gene in adult and wild puffer fish was EF1-α>β-action>18S rRDA>GAPDH. Among the three developmental stages of puffer fish ( 3 months, 6 months and 12 months) , EF1-αgene was the most stable, while other tissues had different selection of reference genes in different development stages. The results provided an important reference for quantitative analysis of functional gene expression in T. flavidus under different conditions